Unique Functional and Structural Properties of the LRRK2 Protein ATP-binding Pocket

Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson disease. The most common mutation, G2019S, resides in the kinase domain and enhances activity. LRRK2 possesses the unique property of cis-autophosphorylation of its own GTPase domain. Because high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-autophosphorylation and trans-peptide phosphorylation to probe the ATP-binding pocket. We disclose hundreds of commercially available activity-selective LRRK2 kinase inhibitors. Some compounds inhibit cis-autophosphorylation more strongly than trans-peptide phosphorylation, and other compounds inhibit G2019S-LRRK2 more strongly than WT-LRRK2. Through exploitation of structure-activity relationships revealed through high-throughput analyses, we identified a useful probe inhibitor, SRI-29132 (11). SRI-29132 is exquisitely selective for LRRK2 kinase activity and is effective in attenuating proinflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Furthermore, the compound demonstrates excellent potency, is highly blood-brain barrier-permeant, but suffers from rapid first-pass metabolism. Despite the observed selectivity of SRI-29132, docking models highlighted critical interactions with residues conserved in many protein kinases, implying a unique structural configuration for the LRRK2 ATP-binding pocket. Although the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2 homolog from ameba can be mutated to approximate some aspects of the human LRRK2 ATP-binding pocket. Our results provide a rich resource for LRRK2 small molecule inhibitor development. More broadly, our results provide a precedent for the functional interrogation of ATP-binding pockets when traditional approaches to ascertain structure prove difficult.     

Leishmania amazonensis arginase compartmentalization in the glycosome is important for parasite infectivity

In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg(-) L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg(-) mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol-targeted ARG lacking the glycosomal SKL targeting sequence (argΔSKL) restored growth but failed to restore infectivity. Further study showed that the ARGΔSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration.     

New digestive symbiosis in the hydrothermal vent amphipoda Ventiella sulfuris

Ventiella sulfuris Barnard and Ingram, 1990 is the most abundant amphipod species inhabiting the Eastern Pacific Rise (EPR 9°N) vent fields. This vent-endemic species is frequently encountered near colonies of Pompeii worms Alvinella pompejana. V. sulfuris specimens were collected during the oceanographic cruise LADDER II at the Bio9 (9°50.3′ N, 2508 m depth) hydrothermal vent site. Main objectives were to highlight the occurrence of bacterial symbiosis in V. sulfuris and to hypothesise their implications in nutrition. Observations in light and electron microscopy (SEM, TEM) showed that the outer body surface and appendages are free of microorganisms. In contrast, the digestive system revealed two major microbial communities settled in the midgut and in the hindgut. Gut contents showed bacterial traces together with abundant fragments of Alvinellid cuticle and setae, from A. pompejana, suggesting that V. sulfuris could directly feed on Alvinellids and/or on their bacterial epibionts. Molecular analyses based on the 16S rRNA genes revealed the diversity of bacterial communities in the digestive system, of which, the Epsilonproteobacteria phylum, could be considered as one of the major bacterial group. Hypotheses were proposed on their symbiotic features and their implications in V. sulfuris nutrition.